G-coupled receptor showing selective affinity for ATP

ABSTRACT

The present invention relates to an isolated G-protein coupled receptor, nucleic acid sequence encoding the receptor, and host cells capable of expressing the receptor. The invention further comprises methods for detecting the expression of a G-protein coupled receptor, and methods for identifying agonists or antagonists of the receptor. The invention still further encompasses methods for preparing an isolated G-protein coupled receptor.

CROSS-REFERENCE TO RELATED APPLICATION

The present invention is a continuation of U.S. application Ser. No.09/254,783, filed Aug. 16, 1999, now abandoned, which claims priority toPCT/BE98/00108, filed Jul. 9, 1998, now published as WO99/02675, whichclaims priority to EP97870101.9, filed Jul. 9, 1997. The contents ofeach priority application is incorporated by reference herein in itsentirety.

OBJECT OF THE PRESENT INVENTION

The present invention concerns a new G protein-coupled receptor havingselective affinity for ATP and the nucleic acid molecule encoding saidreceptor, vectors comprising said nucleic acid molecule, cellstransformed by said vector, antibodies directed against said receptor,nucleic acid probes directed against said nucleic acid molecule,pharmaceutical compositions comprising said products and non humantransgenic animals expressing the receptor according to the invention orthe nucleic acid molecule according to said receptor.

BACKGROUND OF THE INVENTION

An impressive number of P2 receptors subtypes has been cloned since1993. A new molecular nomenclature has then been created in which Gprotein-coupled P2 receptors have been named P2Y while P2 receptorshaving an intrinsic ion channel activity have been named P2X. The P2Yfamily encompasses selective purinoceptors (the P2Y₁ receptor activatedby ATP and ADP), nucleotide receptors responsive to both adenine anduracil nucleotides (P2Y₂ receptor: activated equipotentally by ATP andUTP) and pyrimidinoceptors (the P2Y₃ and P2Y₆ receptors activated byUDP; the P2Y₄ receptor: activated by UTP). The P2Y₅ and P2Y₇ receptorsdisplay limited homologies with the other members of the P2Y family.They have been included in this family especially on the basis ofradioligand binding studies showing affinities for adenine nucleotides(1–18).

SUMMARY OF THE INVENTION

The present invention concerns a new receptor having the amino acidssequence of FIG. 1 or any receptor which presents more than 50%,preferably more than 70%, more preferably more than 85%, morespecifically more than 95% homology with the amino acids sequence ofFIG. 1.

The present invention concerns also the receptor having at least theamino acids sequence of FIG. 1 or a portion thereof, preferably an aminoacids sequence wherein the large extracellular part (the NH2 portion of450 amino acids) has been truncated at the (♦) (as represented onFIG. 1) or active parts of said portion such as the sixth and seventhtransmembrane domains comprising the amino acids: His⁶⁸⁶, Arg⁶⁸⁹ andArg⁷²⁸.

The present invention is also related to said NH₂ portion of 450 aminoacids sequence, including peptides reproducing or mimicking a portion ofthis sequence or of organic molecules sharing the effects of thesepeptides.

Indeed, the inventors have discovered that either the whole receptorhaving the amino acids sequence of FIG. 1 or its portion (preferably theamino acids sequence wherein the large extracellular part of 450 aminoacids has been truncated and starting from (♦) in FIG. 1) seems to havethe same industrial application (said portion will be identifiedhereafter as the P2Y₁₁ receptor or sequence).

The first industrial application of this receptor or its portions is thescreening of agonists and antagonists of said receptor which may haveadvantageous pharmaceutical or diagnostical properties. The secondindustrial application of the receptor according to the invention or ofits portions or of active parts of its portions is the identification ofpatients who may present genetic disorders induced by an inactivereceptor or by an inactive portion of said receptor.

According to a preferred embodiment of the present invention, saidreceptor is a human receptor.

ATP seems to be the preferential natural agonist of this receptor: UTP,UDP, AP₄A, AP₆A, AMP and adenosine seem to be unable to stimulate thephosphoinositide pathway or were much less potent that ATP.

Therefore, the invention is also related to a new G-coupled receptor,its portions or active parts of its portions having a selective affinityfor ATP. “A selective affinity for ATP” means that ATP is able to inducethe formation of a functional response (preferably the accumulation ofInositol triphosphate IP₃ and a rise of intracellular Ca²⁺) in a shorttime of incubation with said agonist (preferably in less than 5 min;more preferably less than 1 min) while the other known agonists of P2Y(UTP, UDP, AP₄A, AP₆A, AMP and adenosine) were unable to stimulate saidreceptor or were much less potent than ATP and induce a detectablefunctional response by said receptor.

The present invention is also related to a nucleic acid molecule, suchas a DNA molecule or an RNA molecule, encoding the receptor, itsportions or active parts of its portions according to the invention.

Preferably, said DNA molecule is a cDNA molecule or a genomic DNAmolecule.

Preferably, said nucleic acid molecule has more than 50%, preferablymore than 70%, more preferably more than 85%, more specifically morethan 95% homology with the DNA sequence shown in FIG. 1.

Preferably, the invention is related to a nucleic acid molecule whichhas more than 50%, preferably more than 70%, more preferably more than85%, more specifically more than 95% homology with this DNA sequence(shown in FIG. 1), wherein the DNA sequence encoding the 450 amino acidsof the NH₂ portion were truncated.

The present invention is also related to the vector comprising thenucleic acid molecule according to the invention. Preferably, saidvector is adapted for expression in a cell and comprises the regulatoryelements necessary for expressing the amino acid molecule in said celloperatively linked to the nucleic acid sequence according to theinvention as to permit expression thereof.

Preferably, said cell is selected from the group consisting of bacterialcells, yeast cells, insect cells or mammalian cells. The vectoraccording to the invention is a plasmid or a virus, preferably abaculovirus, an adenovirus or a Semliki Forest virus.

The present invention concerns also the cell transformed by the vectoraccording to the invention, said cell is preferably non-neuronal inorigin and is selected from the group consisting of a COS-7 cell, a CHOcell, an LM(tk−) cell, an NIH-3T3 cell or a 1321N1 astrocytoma cell.

The present invention is also related to a nucleic acid probe comprisingthe nucleic acid molecule according to the invention, of at least 15nucleotides capable of specifically hybridising with a unique sequenceincluded in the sequence of the nucleic acid molecule encoding thereceptor according to the invention. Said nucleic acid probe may be aDNA or an RNA molecule.

The invention concerns also an antisense oligonucleotide having asequence capable of specifically hybridising to an mRNA moleculeencoding the receptor according to the invention so as to preventtranslation of said mRNA molecule or an antisense oligonucleotide havinga sequence capable of specifically hybridising to the cDNA moleculeencoding the receptor according to the invention.

Said antisense oligonucleotide may comprise chemical analogs ofnucleotide or substances which inactivate mRNA, or be included in an RNAmolecule endowed with ribozyme activity.

Another aspect of the present invention concerns a ligand (preferably anantibody) other than known molecules, especially the ATP, capable ofbinding to the receptor according to the invention and an anti-ligand(preferably also an antibody) capable of competitively inhibiting thebinding of said ligand to the receptor according to the invention.

Preferably, said antibody is a monoclonal antibody directed to anepitope of the receptor according to the invention and present on thesurface of a cell expressing said receptor.

The invention concerns also the pharmaceutical composition comprising aneffective amount of oligonucleotide according to the invention,effective to decrease the activity of said receptor by passing through acell membrane and binding specifically with mRNA encoding the receptoraccording to the invention in the cell so as to prevent its translation.The pharmaceutical composition comprises also a pharmaceuticallyacceptable carrier capable of passing through said cell membrane.

Preferably, in said pharmaceutical composition, the oligonucleotide iscoupled to a substance, such as a ribozyme, which inactivates mRNA.

Preferably, the pharmaceutically acceptable carrier comprises astructure which binds to a receptor on a cell capable of being taken upby cell after binding to the structure. The structure of thepharmaceutically acceptable carrier in said pharmaceutical compositionis capable of binding to a receptor which is specific for a selectedcell type.

Preferably, said pharmaceutical composition comprises an amount of theantibody according to the invention effective to block the binding of aligand to the receptor according to the invention and a pharmaceuticallyacceptable carrier.

The present invention concerns also a transgenic non human mammaloverexpressing (or expressing ectopically) the nucleic acid moleculeencoding the receptor according to the invention.

The present invention also concerns a transgenic non human mammalcomprising a homologous recombination knockout of the native receptoraccording to the invention.

According to a preferred embodiment of the invention, the transgenic nonhuman, mammal whose genome comprises antisense nucleic acidcomplementary to the nucleic acid according to the invention is soplaced as to be transcripted into antisense mRNA which is complementaryto the mRNA encoding the receptor according to the invention and whichhybridises to mRNA encoding said receptor, thereby reducing itstranslation. Preferably, the transgenic non human mammal according tothe invention comprises a nucleic acid molecule encoding the receptoraccording to the invention and comprises additionally an induciblepromoter or a tissue specific regulatory element.

Preferably, the transgenic non human mammal is a mouse.

The invention relates also to a method for determining whether a ligandas an agonist or an antagonist of the receptor according to theinvention can be specifically bound to said receptor; said methodcomprising the steps of contacting a cell or a cell extract from cellstransfected with a vector according to the invention and expressing thenucleic acid molecule encoding said receptor, possibly isolating amembrane fraction from the cell extract, contacting the ligand with themembrane fraction or with the cell under conditions permitting bindingof said ligand to the receptor and detecting, possibly by means of abioassay such as a modification in the second messenger concentration ora modification in the cellular metabolism (preferably determined by theacidification rate of the culture medium), an increase in the receptoractivity, thereby determining whether the ligand binds to the receptor,possibly as an agonist or as an antagonist of said receptor.

Preferably, the second messenger assay comprises measurement ofintracellular cAMP, intracellular inositol phosphate (IP3),intracellular diacylglycerol (DAG) concentration or intracellularcalcium mobilisation.

Preferably, the cell used in said method is a mammalian cell nonneuronal in origin, such as a COS-7 cell, a CHO cell, a LM(tk−) cell anNIH-3T3 cell or 1321N1 cell. In said method, the ligand is notpreviously known.

The invention is also related to the ligand isolated and detected by anyof the preceding methods.

The present invention concerns also the pharmaceutical composition whichcomprises an effective amount of an agonist or an antagonist of thereceptor according to the invention, effective to reduce the activity ofsaid receptor and a pharmaceutically acceptable carrier.

The P2Y₁₁ transcripts (obtained from the nucleotidic sequence startingfrom (♦) in FIG. 1) are detectable in HL-60 human leukaemia cells.Expression of P2Y₁₁ receptor mRNA is increased by agents (ripnoic acid,DMSO) known to induce the granulocytic differenciations of HL-60 cells.However, the P2Y₁₁ transcripts could not be detected in matureneutrophils. Therefore, a first industrial application of the productaccording to the invention is the diagnostic of leukaemia, preferably byNorthern blot analysis using the nucleotidic sequence encoding the P2Y₁₁receptor according to the invention.

The present invention is also related to a diagnostic device or kitcomprising the elements for the diagnostic of specific leukaemia,preferably HL-60 human leukaemia, comprising the receptor according tothe invention, the nucleic acid sequence encoding said receptor, anucleic acid probe comprising the nucleic acid molecule according to theinvention of at least 15 nucleotides capable of specifically hybridisingwith a unique sequence included in the sequence of the nucleic acidmolecule encoding the receptor according to the invention, such as anantisense oligonucleotide or a ligand such as an antibody, preferably amonoclonal antibody, capable of binding or competitively inhibiting thebinding of a ligand to the receptor according to the invention. Saiddiagnostic device or kit could be used for the specific diagnostic orfor the monitoring of the evolution of tumoral cells, especiallyleukaemia HL-60 cells.

Therefore, the previously described methods may be used for thescreening of drugs (having advantageously anti-tumoral properties) whichspecifically bind to the receptor according to the invention.

Another industrial application of the present invention is related tothe use of said drugs, preferably ligands or anti-ligands according tothe invention, for the prevention and/or the treatment of specificdiseases such as neutropenie or agranulocytose infections or cancer.

The invention is also related to the drugs isolated and detected by anyof these methods.

The present invention concerns also a pharmaceutical compositioncomprising said drugs and a pharmaceutically acceptable carrier.

The invention is also related to a method of detecting expression of areceptor according to the invention by detecting the presence of mRNAcoding for a receptor, which comprises obtaining total RNA or total mRNAfrom the cell and contacting the RNA or mRNA so obtained with thenucleic acid probe according to the invention under hybridisingconditions and detecting the presence of mRMA hybridised to the probe,thereby detecting the expression of the receptor by the cell.

The hybridisation conditions above-described are preferably standardstringent conditions as described by Sambrook et al. (§9.47-9.51 inMolecular Cloning: A Laboratory Manual, Cold Spring Harbour, LaboratoryPress, New York (1989)).

The present invention concerns also a method for diagnosing apredisposition to a disorder associated with the activity of thereceptor according to the invention. Said method comprises:

-   -   a) obtaining nucleic acid molecules of subjects suffering from        said disorder;    -   b) performing a restriction digest of said nucleic acid        molecules with a panel of restriction enzymes;    -   c) electrophoretically separating the resulting nucleic acid        fragments on a sized gel;    -   d) contacting the resulting gel with a nucleic acid probe        capable of specifically hybridising to said nucleic acid        molecule and labelled with a detectable marker;    -   e) detecting labelled bands which have hybridised to the said        nucleic acid molecule labelled with a detectable marker to        create a unique band pattern specific to subjects suffering from        said disorder;    -   f) preparing nucleic acid molecules obtained for diagnosis by        step a-c; and    -   g) comparing the unique band pattern specific to the nucleic        acid molecule of subjects suffering from the disorder from step        e and the nucleic acid molecule obtained for diagnosis from step        f to determine whether the patterns are the same or different        and to diagnose thereby predisposition to the disorder if the        patterns are the same.

A last aspect of the present invention concerns a method of preparingthe receptor according to the invention, which comprises:

-   -   a) constructing a vector adapted for expression in a cell which        comprises the regulatory elements necessary for the expression        of nucleic acid molecules in the cell operatively linked to        nucleic acid molecule encoding said receptor so as to permit        expression thereof, wherein the cell is selected from the group        consisting of bacterial cells, yeast cells, insect cells and        mammalian cells;    -   b) inserting the vector of step a in a suitable host cell;    -   c) incubating the cell of step b under conditions allowing the        expression of the receptor according to the invention;    -   d) recovering the receptor so obtained; and    -   e) purifying the receptor so recovered, thereby preparing an        isolated receptor according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the nucleotide and deduced amino acid sequence of thenew human P2Y receptor. The putative phosphorylation sites by proteinkinase C or by calmodulin-dependent protein kinases are indicatedrespectively by a black circle (●) or an unside down black triangle (▾).The potential N-glycosylation site is indicated by a black square (▪).

FIG. 2 represents dendrogram of the structural relatedness of the P2Y₁₁receptor with the other P2Y subtypes. The plot was constructed using themultiple sequence alignment program Pileup of the GCG package. TheP2Y₅-like published sequence (18) is identical to the P2Y₉ sequencesubmitted to the GenBank/EMBL Data Bank.

FIG. 3 represents Northern blot analysis of P2Y₁₁ messenger expression.Each lane of the MTN blot contains 2 μg of polyA⁺ RNA from several humantissues. Each lane of the HL-60 blot contains 10 μg of total RNA fromdifferentiated or undifferentiated HL-60 cells. Hybridization with theprobe was performed as described under Materials and Methods. Thepictures of the MTN II blot and the HL-60 blot were obtainedrespectively, from an autoradiography and from a Phosphorlmager SI(Molecular Dynamics). The 2 kb-length P2Y₁₁ transcripts are indicated bya black arrow.

FIG. 4 represents concentration-action curves of several nucleotides onIP₃ and cAMP accumulation in cells transfected with the P2Y₁₁ receptor.1321N1 and CHO-K1 transfected cells were assayed for the accumulationof, respectively, IP₃ (A) or cAMP (B) in response to variousconcentrations of the following nucleotides: ATP, 2MeSATP, ADP and2MeSADP. Incubation times were 30 s for IP₃ measurements and 15 min forcAMP assays. The data represent the means ±S.D. of triplicateexperimental points and are representative of two independentexperiments.

DESCRIPTION OF A PREFERRED EMBODIMENT OF THE PRESENT INVENTIONExperimental Procedures

Materials

Trypsin was from Flow Laboratories (Bioggio, Switzerland). Culturemedia, G418, fetal calf serum (FCS), restriction enzymes and Taqpolymerase were purchased from GIBCO ERL (Grand Island, N.Y.). Theradioactive products myo-D-[2-³H]inositol (17.7 Ci/mmol) and [α³²P]ATP(800 Ci/mmol) were from Amersham (Ghent, Belgium). Dowex AG1X8 (formateform) was from Bio-Rad Laboratories (Richmond, Calif.) ATP, ADP, AMP,adenosine, UTP, UDP, AP₄A, AP₆A, all-trans retinoic acid (RA) and12-O-tetradecanoylphorbol-3-acetate (TPA) were obtained from SigmaChemical Co. (St. Louis, Mo.). 2-methylthio-ATP (2MeSATP),2-methylthio-ADP, (2MeSADP) and 8 (p-sulfophenyl) theophylline were fromResearch Biochemicals International (Natick, Mass.). Forskolin waspurchased from Calbiochem (Bierges, Belgium). Indomethacin and dimethylsulfoxide (DMSO) were from Merck (Netherlands). Rolipram was obtainedfrom the Laboratoires Jacques Logeais (Trappes, France). The HL-60 humancell line was obtained from the American Type Culture Collection(Rockville, USA). The human genomic DNA library was from Stratagene (LaJolla, Calif.). pEFIN3 is an expression vector obtained from Euroscreen(Brussels, Belgium). Multiple Human Tissues Northern blot (MTN) werefrom Clontech (Palo Alto, Calif.).

Cloning and Sequencing

A human placenta cDNA library was screened at moderate stringency withan [α³²P] dATP labelled P2Y₄ receptor probe corresponding to a partialsequence covering the third to the seventh transmembrane segments. Threeoverlapping clones encoding a new G protein-coupled receptor wereisolated, but did not contain the 3′ end of the coding region. A humangenomic DNA library was then screened with this partial sequence toobtain the complete sequence of this new receptor. The hybridizationconditions for screening the two libraries were 6× SSC (1× SSC: 0.15 MNaCl, 0.015 M sodium citrate) and 40% formamide at 42° C. for 14 hoursand the final washing conditions were 0.5× SSC, 0.1% SDS at 60° C. Fourgenomic clones were purified and shown to contain the 3′ end of the openreading frame missing in the cDNA clones. The sequence was obtained onboth strands after subcloning of overlapping restriction fragments inM13mp18 and M13mp19 using the Sanger dideoxy nucleotide chaintermination method.

Northern Blot Analysis

Two blots of human organs (MTN I and MTN II: 2 μg polyA⁺ RNA/lane) and ablot containing total RNA from differentiated and undifferentiated HL-60cells (10 μg of total RNA/lane) were hybridized with a probecorresponding to the new receptor in order to characterize its tissuedistribution. The HL-60 cells were maintained in RPMI 1640 supplementedwith 10% FCS, 5 mM L-glutamine, 50 U/ml penicillin and 50 μg/mlstreptomycin at 37° C. with 5% CO₂. The HL-60 cells were incubatedduring six days with or without 1 μM retinoic acid or 1.25% DMSO orduring eight hours with 25 nM TPA. The RNA from the differentiated orundifferentiated HL-60 cells was prepared with the RNeasy kit (Quiagen).The blots were prehybridized 8 hours at 42° C. in a 50% formamide, 2%SDS solution and hybridized for 18 hours in the same solutionsupplemented with the [α³²p] labelled probe. The final washingconditions were 0.1× SSC and 0.1% SDS at 55° C. The blots were exposedduring twelve days and visualized as an autoradiography or using thePhosphorlmager SI (Molecular Dynamics).

Cell Culture and Transfection

The complete sequence of the new receptor according to the invention wassubcloned between the Hind III and Nhe I sites of the bicistronic pEFIN3expression vector. 1321N1 and CHO-K1 cells were transfected with therecombinant pEFIN3 plasmid or with the plasmid alone using the calciumphosphate precipitation method as described (19). The transfected cellswere selected with 400 μg/ml G418 in complete medium (10% FCS, 100units/ml penicillin, 100 μg/ml streptomycin and 2.5 μg/ml amphotericin Bin Duibecco's modified Eagle'medium (DMEM)) two days after transfectionand maintained in the same medium (10).

Inositol Phosphates (IP) Measurement

1321N1 cells were labelled for 24 hours with 10 mCi/ml [³H] inositol ininositol free DMEM containing 5% FCS, antibiotics, amphotericin, sodiumpyruvate and 400 μg/ml G418. Cells were washed twice with Krebs-RingerHepes (KRH) buffer of the following composition (124 mM NaCl, 5 mM KCl,1.25 mM MgSO₄, 1.45 mM CaCI₂, 1.25 mM KH₂PO₄, 25 mM Hepes (pH:7.4) and 8mM glucose) and incubated in the same medium for 30 min. The cells werethen challenged by various nucleotides for 30 s. The incubation wasstopped by the addition of an ice cold 3% perchioric acid solution. IPwere extracted and separated on Dowex columns as previously described(20).

Cyclic AMP Measurements

Stably transfected CHO-K1 or 1321N1 cell lines were spread on Petridishes (150.000 cells per dish) and cultured in Ham's F12 or DMEM mediumcontaining 10%, FCS, antibiotics, amphotericin, sodium pyruvate and 400μg/ml G418. Cells were preincubated for 30 min in KRH buffer withRolipram (25 μM) and incubated for different times in the presence ofthe agonists (15 min in most experiments). The incubation was stopped bythe addition of 1 ml HCl 0.1 M. The incubation medium was dried up,resuspended in water and diluted as required. Cyclic AMP was quantifiedby radioimmunoassay after acetylation as previously described (21).

Results

Cloning and Sequencing

A human cDNA placenta library was screened at moderate stringency with ahuman P2Y₄ probe. Nine clones which hybridized weakly with the P2Y₄probe were obtained, purified and analyzed. Six of them corresponded tothe sequence of the P2Y₆ receptor (10) while three overlapping clonescorresponded to a partial sequence encoding a new G protein-coupledreceptor, displaying about 30% identity with the other P2Y receptors.The partial open reading frame started with an ATG-codon in a Kozakconsensus but the 3′ end was missing in all three cDNA clones. TheInventors screened a human genomic DNA library using this partialsequence as a probe. Four overlapping genomic clones were obtained.Mapping of the coding sequence and partial sequencing allowed todetermine that the gene encoding the new receptor contains an introninterrupting the coding sequence at the 5′ end of the gene. This intronseparates the three first codons from the rest of the coding sequence.Beside these first codons, the four genomic clones contained thecomplete open reading frame including the 3′ end missing in the cDNAclones. The full open reading frame appeared as 1113 base pairs (bp)long and encoded a protein of 371 amino acids containing one potentialsite for N-linked glycosylation and two potential sites forphosphorylation by protein kinase C or calmodulin-dependent proteinkinases (FIG. 1). The new receptor, provisionally named P2Y₁₁, displayssignificant homologies with the other P2Y receptors (FIG. 2). Inparticular, 33% and 28% amino acid identity were observed respectivelywith the human P2Y₁ and P2Y₂ receptors.

Tissue Distribution of the New Receptor

The tissue distribution of the new receptor transcripts was investigatedby Northern blotting (FIG. 3) by using a probe corresponding to apartial sequence encoding transmembrane segments 3 to 7. The strongestsignal was observed for human spleen and corresponded to a 2 kilobase(kb)-length messenger RNA (MTN II). A weaker signal was observed insmall intestine (MTN II). All the lanes in MTN I (heart, brain,placenta, lung, liver, skeletal muscle, kidney, pancreas) were negative.The Inventors also detected specific 2 kb-length transcripts in HL-60cells. The signal was very weak in the undifferentiated HL-60 cells butincreased when the cells had been treated with retinoic acid or DMSO. Noincrease was observed when the HL-60 cells were stimulated with TPA. Aweak non-specific hybridization with 18S mRNA was observed. These datawere confirmed with a non-overlapping probe corresponding to the first300 bp of the coding region, presenting limited homologies with theother P2Y subtypes.

Functional Expression of the New Receptor in 1321N1 Astrocytoma Cells

The complete sequence of the new receptor was introduced in the pEFIN3expression vector in order to transfect the 1321N1 astrocytoma cellline, used previously to characterize several P2Y subtypes (6, 10, 12).The pool of G418-resistant clones was tested for its functional responseto several nucleotides. ATP (100 μM) induced a strong inositoltrisphosphate (IP₃) accumulation in cells transfected with therecombinant plasmid, whereas ADP, AMP, adenosine, UTP, UDP, AP₄A andAP₆A were inactive at the same concentration. All nucleotides weretotally inactive on the cells transfected with the vector alone. We thentested ATP, 2MeSATP, ADP and 2MeSADP in a large range of concentrations.As shown in FIG. 4A, ATP was the most potent agonist (EC₅₀ATP=38±7 μM;EC₅₀ 2MeSATP=118±15 μM; means±range of two independent experiments). Theeffect of ADP and 2MeSADP were minimal. Pertussis toxin (50 ng/ml; 24 hpretreatment) had no effect on the ATP response, whereas a lowerconcentration of pertussis toxin was previously shown to abolish theresponse to UTP in P2Y₄ transfected 1321N1 astrocytoma cells (22). Aresponse to ATP (10 μM) was also obtained following [Ca²⁺]₁,measurements performed on the 1321N1 transfected cells while ADP wasinactive at this concentration.

Functional Expression of the New Receptor in CHO-K1 cells

The 1321N1 cells transfected with the new receptor displayed a strongcAMP increase in response to ATP. A much lower but significantendogeneous response due to the degradation of adenine nucleotides intoadenosine was also obtained in the 1321N1 cells transfected with thevector alone. The CHO-K1 cells express an endogeneous P2Y₂ receptorcoupled to the phosphoinositide pathway (23) but do not possessadenosine receptors coupled to adenylyl cyclase. We therefore usedCHO-K1 cells in order to characterize the coupling of the new receptorto the cAMP pathway. A pool of G418 resistant CHO-K1 clones was firsttested for its response to several nucleotides at a concentration of 100μM. ATP was able to induce a strong increase in the cAMP content,whereas it was inactive on cells transfected with the vector alone. ADP,AMP, adenosine, UTP and UDP were completely inactive.Concentration-action curves were established for ATP, 2MeSATP, ADP and2MeSADP (FIG. 4B). The rank order of potency was the same as in theinositol phosphate study on 1321N1 cells. The curves were obtained after15 min of stimulation by the agonists; however a significant cAMPresponse to ATP was already obtained after 2 min of stimulation. Theresponse to ATP (30 μM) was inhibited neither by indomethacin (10 μg/ml,present from 30 minutes before the stimulation and readded in thestimulation medium) nor by 8 (p-sulfophenyl) theophylline (100 μM).

The receptor according to the invention presents some structuralpeculiarities which differentiate it from some other P2Y subtypes.Concerning its gene structure, the coding sequence is interrupted by anintron. Comparison between the cDNA and the genomic DNA sequences hasclearly demonstrated the absence of intron in the coding region of thehuman P2Y₁ receptor (24, 25), the rat P2Y₂ receptor (26) and the ratP2Y₆ receptor (11). In terms of protein structure, the second and thirdextracellular loops are significantly longer than those of the other P2Yreceptors. The homology with the other subtypes is relatively weak(about 30%). The closest G-coupled receptor is the human P2Y₁ receptor(33%) which is also a receptor responsive to adenine nucleotides (3, 4).Mutagenesis experiments with the P2Y₂ receptor have identified threepositively charged amino acids in the sixth and seventh transmembranedomains (His²⁶², Arg²⁶⁵ and Arg²⁹²), which play a crucial role innucleotide binding (presumably by neutralizing the negative charge ofthe phosphate groups) (27). These three residues are conserved in thisnew receptor.

So far, eight P2Y receptor subtypes are described in the literature(P2Y₁–22Y₈). In addition, two sequences related to the P2Y₅ receptor andnamed P2Y₉ and P2Y₁₀, have been recently submitted to the GenBank/EMBLData Bank. The P2Y₉ sequence is identical to that recently publishedunder the name “P2Y₅-like” (18). Therefore the new receptor described inthis paper might be called P2Y₁₁. However, it is already clear that thenomenclature needs a revision. It was recently demonstrated that theP2Y₇ receptor is actually a receptor for leukotriene B₄ (16) and thereis no functional evidence that the P2Y₅ and related receptors (P2Y₅-likeor P2Y₉, P2Y₁₀) are nucleotide receptors (17, 18).

Among the sixteen human organs tested by Northern blotting, P2Y₁₁transcripts of 2 kb size were only detectable in spleen, and with lowerintensity in small intestine. This distribution is reminiscent of thatof the human P2Y₆ 1.7 kb-messenger. The observation of the expression ofthe P2Y₁₁ receptor in the HL-60 cell line shows that this expression wasstrongly increased following treatment by DMSO or retinoic acid, twoagents known to induce the differentiation of these cells intogranulocytes (28). On the contrary, TPA, which is known to induce themonocytic differentiation of the HL-60 cells (29), did not stimulate theexpression of the P2Y₁₁, receptor. The confirmation of these data with asecond probe of the P2Y₁₁ cDNA, that shares little similarity with otherP2Y sequences, excludes possible cross-hybridization with another P2Yreceptor transcript. In view of the Northern blots results, it istempting to speculate that the P2Y₁₁ receptor is involved in therecently described accumulation of CAMP in ATP-stimulated HL-60 cells(30).

Among the P2Y receptors, the P2Y₁₁ subtype has the unique property toactivate both the phosphoinositide and the cAMP pathways. Other clonedP2Y receptors are coupled to phospholipase C exclusively. The rank orderof potency of agonists was the same for the two pathways. ATP wasclearly much more potent than ADP. This difference may be evenunderestimated as a result of low level ATP contamination in ADPpreparation or conversion of ADP into ATP during assays (4, 11). On theother hand, 2MeSATP had the same maximal effect than ATP but presented alower potency, while 2MeSADP, a potent activator of the P2Y₁ and P_(2T)subtypes (4), was almost inactive. The EC₅₀ values were comparable tothose obtained in the study concerning the effects of extracellularnucleotides on the cAMP accumulation in the HL-60 cells (30).

Stimulatory effects of adenine nucleotides on the cAMP pathway have beendescribed in different cell types (31, 32). In most cases, thestimulatory effect of nucleotides was inhibited by xanthines. These datasuffer from the fact that it is difficult to exclude that the effect ofadenine nucleotides is mediated by their degradation into adenosine dueto the ubiquitous presence of ectonucleotidases expressed at the cellsurface. The cAMP study has been performed with CHO-K1 cells to avoidthe endogeneous cAMP response to adenosine in the astrocytoma cell line.Neither in untransfected CHO-K1 cells nor in P2Y₁₁-transfected CHO-K1cells did adenosine increase cAMP accumulation. Furthermore the cAMPresponse to ATP was insensitive to xanthine inhibition. It was alsoinsensitive to indomethacin, indicating that is not mediated by therelease of prostaglandins. It is unlikely that the cAMP response wouldbe an indirect consequence of the calcium response since the use of ATP,which activates the phosphoinositide pathway by the activation of P2Y₂endogeneous receptors, or the use of calcium ionophores in the CHO-K1cells failed to stimulate cAMP accumulation (33). Therefore these dataconstitute the first strong evidence that a P₂ receptor can be coupledto the stimulation of adenylyl cyclase.

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1. An isolated nucleic acid molecule encoding a receptor comprising anamino acid sequence that is at least 95% identical to amino acidresidues 424–795 of SEQ ID NO:
 1. 2. The isolated nucleic acid moleculeaccording to claim 1, wherein the isolated nucleic acid molecule is aDNA or RNA molecule.
 3. The isolated nucleic acid molecule according toclaim 1, wherein the isolated nucleic acid molecule is a cDNA moleculeor a genomic DNA molecule.
 4. An isolated nucleic acid molecule havingat least 95% identity to a nucleic acid molecule consisting of nucleicacid residues 1309–2424 of the nucleic acid sequence of SEQ ID) NO: 2.5. A vector comprising the isolated nucleic acid molecule according toclaim 1 or
 4. 6. An isolated cell comprising the vector according toclaim 5.